PROJECT SUMMARY A patient's own T cells can be modified using gene therapy technology to express receptors, termed chimeric antigen receptors or CARs, which allow these immune T cells to recognize proteins on the tumor cell surface and in turn allow these CAR modified T cells to recognize and kill the patient's own tumor cells. To this end, the ability to generate patient T cells to recognize ovarian cancer cells is a promising and novel approach to treating very poor prognosis patients with advanced or relapsed ovarian carcinomas. The rationale for using patient T cells modified to recognize tumor cells is based on clinical observations demonstrating a correlation between tumor progression and the presence of the patient's own endogenous T cells within the tumor which suggests that a patient's own immune response to the cancer has clinical benefit. Therefore it is reasonable to propose that if we could generate in the laboratory a large population of tumor specific T cells, upon reinfusion, these T cells should be able to significantly kill off a large number of these ovarian cancer tumor cells. To this end, application of this approach requires that we identify a T cell target on the surface of the ovarian cancer tumor cell and subsequently generate a CAR which recognizes this target. MUC-16ecto is a novel identified target derived from MUC-16, a glycoprotein expressed on a majority of ovarian carcinomas, which is significantly retained on the tumor cell surface, making this an attractive target for genetically modified CAR T cells. To this end, we have recently generated just such a CAR, termed 4H11-28z, which recognizes the MUC-16ecto target over-expressed on most peritoneal and ovarian cancer tumor cells. However, while the generation of these tumor specific CAR T cells in the laboratory is required for this immune-based approach to ovarian cancer, it alone is not likely to be sufficient to obtain optimal anti-tumor effects when treating patients. Specifically, successful clinical application of this T cell therapy approach will require further modifications ogf the T cell to overcome tumor mediated immune suppression of these tumor targeted CAR T cells. To this end, we have generated and validated a tri-cistronic gamma-retrovial vector containing the 4H11-28z CAR specific to the retained MUC-16ecto antigen as well as genes encoding for the pro-inflammatory flexi IL-12 (fIL- 12)cytokine designed to enhance CAR T cell antitumor efficacy, modulation of the immunosuppressive tumor microenvironment and recruitment of the endogenous anti-tumor response. Additionally CAR T cells will be modified to express a truncated epidermal growth factor receptor (EGFRt) which will be utilized as a suicide vector for elimination of these CAR T cells with the EGFR specific MAb cetuximab in the context of unforeseen toxicities. In Aim 1 of this project we will conduct a phase I dose escalation clinical trial treating relapsed MUC- 16ecto+ peritoneal/ovarian tumors with 4H11-28z/fIL-12/EGFRt autologous T cells. We will assess safety and tolerability of this therapy and as secondary endpoints we will assess response to therapy as well as the impact of therapy on CAR T cell persistence and modulation of the ovarian tumor microenvironment. In Aim 2 of this project we will validate the clinical relevance of an immune competent orthotopic murine model of ovarian cancer based on clinical trial outcomes in Aim 1 and further investigate whether the additional therapy through checkpoint blockade with a PD-1 targeted an antibody may enhance the anti-tumor efficacy of CAR T cells and will further explore a clinically relevant approach to in vivo CAR T cell imaging. Collectively we anticipate that positive outcomes of the pre-clinical studies proposed in Aim 2 of the project (enhanced antitumor efficacy with PD-1 checkpoint blockade, as well as meaningful imagining of CAR T cells utilizing clinically applicable approaches) will be applied to the ongoing clinical trial proposed in Aim 1 of this application through an ammendment of the clinical protocol proposing to treat additional cohorts of patients.